Carboxyl-Modified Amino Acids and Peptides as Protease Inhibitors

Department of Medicinal Chemistry, Uniuersity of Kansas, Lawrence, Kansas ... Chemistry, Victorian College of Pharmacy, Parkville, Victoria, Australia...
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J. Med. Chem. 1986,29, 104-111

104

Carboxyl-Modified Amino Acids and Peptides as Protease Inhibitors Stewart A. Thompson: Peter R. Andrews,t and Robert P. Hanzlik*' Department of Medicinal Chemistry, Uniuersity of Kansas, Lawrence, Kansas 66045, and School of Pharmaceutical Chemistry, Victorian College of Pharmacy, Parkville, Victoria, Australia 3052. Received April 16, 1985 Several types of carboxyl-modified amino acids and peptides were prepared in forms having N-terminal modifications (carrier fragments) suitable for one of several representative protease enzymes, and their inhibitory action toward those enzymes were evaluated. The carboxyl modifications (inhibitory units) included (b) CONH2, (c) CSNHz, (d) CN, (e) trans-CH=CHCOzMe, and (f) trans-CH=CHS02Me. The carrier fragments included NHz(PhCHz)CHX (I),AcNH(PhCHz)CHX (2),HzNCH2CONH(PhCH2)CHX(31,and AcNH(PhCHz)CHCONHCH2X(4). Compounds lb, Id, le, and If were competitive inhibitors of both microsomal and cytosolic leucine aminopeptidase (K, = 14.8, 67,61, and 3.7 mM with the former and 14.1,26.4,27.3, and 8.8 mM with the latter, respectively). Neither compound IC nor leucine thioamide had any detectable effect on either enzyme. Compounds 2b-f were also competitive inhibitors toward chymotrypsin (K, = 13.9, 23.0, 5.3,30.8, and 29.4 mM, respectively). While 4b,4c,and 4d were competitive inhibitors of papain (Ki = 4.7, 0.095, and 0.0011 mM, respectively), 4e proved to be an irreversible affinity label (K, = 0.026 mM and k , = 0.0018 s-l). Inactivation of papain by 4e was retarded in the presence of 4d and could not be reversed by dialysis. Similarly 3b and 3d were competitive inhibitors of dipeptidyl aminopeptidase I (DPP-I, EC 3.4.14.1) (K, = 6.2 and 0.0027 mM, respectively), while 3e and 3f were irreversible affinity labels (Ki = 0.22 and 0.18 mM, and kz = 0.015 and 0.010 s-l, respectively). Inhibition of DPP-I by 3d provides only the second example of a cysteine protease which is strongly inhibited by a nitrile analogue of a specific substrate. Further studies are needed to determine the generality and potential utility of this finding. Compounds 3e,3f,and 4e exemplify a new class of specific affinity labels for cysteine proteases whose activity probably derives from irreversible Michael addition of the catalytic cysteine to the activated double bond.

It is becoming increasingly apparent that protease enzymes play critically important roles in initiating, sustaining, or terminating a wide variety of biological processes. Examples include blood coagulation, complement activation, inflammatory and tissue degenerative diseases, tumor metastasis, viral replication, and many others.'V2 The central role of proteolysis in these processes makes the development of specific, selective, nontoxic protease inhibitiors an attractive challenge in drug design. Since the substrate specificity of many physiologically important proteases is often better known than other features of these enzymes, our approach to this problem has been to modify the structure of known specific substrates in the vicinity of the scissile bond in ways that might be expected to interfere, reversibly or not, with the normal catalytic process. This consideration led us recently to synthesize an analogue of a papain substrate containing a uinylogous amino acid residue. This compound proved to be a specific irreversible inactivator of papain, i.e. an affinity label.3 Here we report that related compounds specifically and irreversibly inactivate another thiol protease, dipeptidyl aminopeptidase-I (DPP-I, EC 3.4.14.1), also known as cathepsin C, but not the serine protease chymotrypsin nor the microsomal or cytosolic forms of the metalloprotease leucine aminopeptidase. We also report on the inhibitory effects of thioamide and nitrile analogues of specific substrates on these enzymes. Design Our purpose was to evaluate the inhibitory potential of functional groups incorporated into substrate analogues by testing them against proteases representing several different classes, while at the same time minimizing the amount of swthesis required. To meet this objective we selected five primary target enzymes which were commercially available and well characterized: (1) chymotrypsin (CT), a serine endoprotease preferring aromatic residues at PI; (2) microsomal and (3) cytosolic leucine aminopeptidase (LAF-M and LAP-C), metalloproteases preferring unblocked N-terminal hydrophobic residues such as leucine or phenylalanine; (4) dipeptidyl amino+ University of Kansas. *Victorian College of Pharmacy.

Scheme I

1 (LAP)

2(chymotrysm)

II

3 (DPP-I)

0

4 (papain)

A. Representative substrates: la-4a. X = CONHC6H4N02 B.Inhibitors: b: X=CONHz:c. X-CSNHz;d-CN: e, X = frcms-CH=CHCOOMe:

f , X =trans-CH=CHS02Me Ph\

AcNH

5d

8

6. R = C H X H 2 7 . R = CHzCHzC02Me

peptidase I (DPP-I, also called cathepsin C), a cysteine protease which cleaves dipeptides from proteins with unblocked N-termini and prefers hydrophobic residues at PI; and (5) papain, a cysteine endoprotease which prefers aromatic residues at P,. A further consideration in the choice of these enzymes was the availability4 of crystal structures for chymotrypsin and papain, which would be useful for molecular graphics analysis of enzyme-inhibitor interactions. Given the specificity of the above enzymes, the synthetic requirements could be streamlined as follows (viz. Scheme I). Carboxyl-modified phenylalanine analogues 1b-f would be directly suitable for studies with LAP; N-terminal acetylation would render them suitable for chymotrypsin (2b-f), while appending an N-terminal glycine would ready them for DPP-I (3b-e). Comparable carboxyl-modified (1) Reich, E.; Rifkin, D. B.; Shaw, E., Eds. "Proteases and Bio-

logical Control"; Cold Spring Harbor Laboratory: Cold Spring, New York, 1975. (2) Berlin, R.D.; Herrmann, H.; Lepow, I. H.; Tanzer, J. M., Eds. "Molecular Basis of Biological Degenerative Processes"; Academic Press: New York, 1978. (3) Hanzlik, R. P.; Thompson, S. A. J. Med. Chem. 1984,27,711. (4) Protein Data Bank. Bernstein, F. C.; Koetzle, T. F.; Williams, G. B.; Meyer, E. F., Jr.; Brice, M. D.; Rodgers, J. R.; Kennard, 0.;Shimanouchi, T.; Tasumi, M. J. Mol. Biol. 1977,112, 535.

0022-2623/86/1829~0104$01.50/0 0 1985 American Chemical Society

Carboxyl-Modified Amino Acids and Peptides

glycine derivatives 4b-f would be suitable for papain. The amide analogues lb-4b would serve as peptide-like alternate-substrate inhibitors and would thus provide a standard of comparison against which to judge the other types of inhibitors for a given enzyme. Results Synthesis. As indicated above, carboxyl-modified derivatives of L-phenylalanine (lb-f) were synthesized for LAP and derivatized further at the N-terminus for chymotrypsin and DPP-I. The Boc derivative of L-lb (BocL-PheCONHz)6was dehydrated with trifluoroacetic anhydride (TFAA) and deprotected with trifluoroacetic acid (TFA) to give Id, isolated as its methanesulfonate salt, in 49% overall yield. Treatment of Boc-L-lb with Lawesson's reagent6 in hexamethylphosphoric triamide (HMPA) formed Boc-lc in 70% yield, deprotection of which with TFA gave IC as a TFA salt in 63% overall yield. For the synthesis of Michael acceptors le and If, BocL-PheCOOMe was converted to Boc-PheCHO by reduction with diisobutylaluminum hydride (Dibal) as described7by Rich et al. and condensed with (Et0)2P(0)CHXNa(X = COOMe or SO2Me)in THF to form the C=C bond, giving Boc-le and Boc-lf, respectively. NMR analysis indicated that only the trans isomers were present, based on the coupling constant of 16 Hz observed in both cases for the vinylic protons. Deprotection with TFA gave le and If, respectively, which were isolated as TFA salts. It is noteworthy that despite the strongly basic conditions of the Horner-Emmons reaction and the general ease of racemization of a-amino aldehydes, le and If and their N-terminal derivatives showed substantial optical activity. However, no attempt was made to determine the enantiomeric purity of these compounds.8 For the synthesis of 4b and 4d, N-Ac-L-Phe was coupled to either glycinamide or aminoacetonitrile by using a standard mixed-anhydride methodag Thioamide 4c was obtained by treating 4d with HzS in pyridine/Et3N. For the synthesis of 4e, Boc-allylamine was ozonized in methanol at -78 OC and the ozonide reduced with MezS to afford Boc-glycinal in 94% yield (crude). The latter was then condensed with (EtO)2P(0)CHNaCOOMein THF, giving truns-Boc-NHCHzCH=CHCO2Me (Boc-5d) in 62% yield; deprotection with TFA gave 5d as the TFA salt in 94% yield. Coupling of 5d, allylamine, and methyl 4aminobutyrate with N-Ac-L-Phe gave 4e, 6, and 7, respectively. Inhibition Studies. Our approach to evaluating new potential inhibitors was to implement a standard photometric assay for the enzyme involved, to determine the K, of the assay substrate for comparison to the reported values, and to evaluate the test compounds via Lineweaver-Burk plots. In most cases competitive inhibition was Our nomenclature of these compounds is baaed on naming the parent amino acid followed by the carboxyl modification. For example, phenylalanine methyl ester would be PheCOOMe (not PheOMe), phenylalaninal would be PheCHO, and N Boc-aminoacetonitrile would be Boc-GlyCN. Clausen, K.; Thorsen, M.; Lawesson, S.-0. Tetrahedron 1981, 37, 3635. Rich, D. H.; Sun, E. T.; Boparai, A. S. J. Org. Chen. 1978,43, 3624.

All of the carboxy-modified phenylalanine derivatives synthesized and tested in this study were prepared from Lphenylalanine. Since there was no reason to expect substantial racemization to occur, particularly in the syntheses leading to amide (b), thioamide (c), or nitrile (d) analogues, no attempt was made to ascertain the optical purities of the products. Chen, F. M. F.; Steinauer, R.; Benoiton, N. L. J. Org. Chem. 1983,48, 2941.

Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1 105

Table I. Michaelis Constants for Assay Substrates K,, mM enzyme substrate obsd lit. chymotrypsin glutaryl-la 0.44 0.65 LAP-M L-Leu-pNA 0.60 0.50 LAP-C

L-Leu-pNA

2.10

DPP-I papain

3a Z-Gly-ONP

3.1 0.0103

0.30 0.52 2.20 1.00 3.3 0.0070 0.0052 0.24 0.88

4a

Table 11. Inhibition Constants enzyme inhib chymotrypsin 2b 2c 2d 2e 2f LAP-M lb IC Id le If LAP-C lb IC Id le If DPP-I 3b 3d 3e 3f papain 4b 4c 4d 4e 6 7

ref 30 33 23 31 23 34 35 36 37 37 12

K,," mM 13.9 23.0 5.3 30.8 29.4 14.8

b 67 61 3.7 14.1 C

26.4 23.7 8.8 6.2 0.0027 0.22 ( k , = 0.015 s-*) 0.18 ( k , = 0.010 9-l) 4.7

0.095 0.0011 0.026 9.2 19.5

(k2

0.0018 8-l)

"All Ki values are for competitive inhibition except those for 3e, 3f, and 4e. *No inhibition with [IC] = 40 mM. cSlight stimulation was observed with [IC] = 5 mM but higher concentrations were not investigated.

indicated, and Ki values were determined from replots of (KmaPP/VmJ vs. [I]. When irreversible inhibition was indicated, pseudo-first-order rate constants (kapp) were determined from the slopes of plots of In (percent remaining activity) vs. time; replots of l / k a p vs. 1/[I] gave the inhibition constants Ki and kz as described by Kitz and Wilson.lo The K, values observed with our assay substrates are collected in Table I, while the kinetic constants for the inhibitors are collected in Table 11. As expected, amides lb-4b proved to be competitive inhibitors of their respective enzymes. In all cases their Ki values are 2-30 times greater than the K, values for the corresponding p-nitroanilides. For many serine and cysteine proteases, the turnover of amide substrates is limited primarily by the rate of enzyme acylation and K, approximates the dissociation constant (K,) for the enzyme-substrate complex. The p-nitrophenyl group is expected to lower K, relative to the amides b through increased hydrophobic interactions and through electron withdrawal which facilitates the acylation step." An extreme example of the latter effect can be seen by comparing the K, values of 4a and the much more reactive (10) Kitz, R.; Wilson, I. J.Bid. Chem. 1962, 237, 3245. (11) Lowe, G.; Yuthavong, Y. Biochem. J. 1971, 124, 117.

106 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1

ester Z-Gly-ONP (Table I). These arguments may or may not apply to LAP-M and LAP-C, since it is not clear whether acyl enzyme intermediates are formed by these enzymes. The nitrile analogues ld-4d showed an interesting pattern of inhibitory effects. The two cysteine proteinases papain and DPP-I were strongly but reversibly inhibited by 4d and 3d,respectively. Toward these enzymes the Ki values of the nitriles were 3-4 X lo3lower than those of the corresponding amides. The Ki value found for 4d (1.1 KM) is in good agreement with the value of 0.73 KM reported earlier by Westerick and Wolfenden.12 Benzamidoacetonitrile (ti), the nitrile analogue of another series of papain substrates, also inhibits papain reversibly, but less dramatically, with a Ki of 0.46 mM.13 As far as we are aware, the inhibition of DPP-I by 3d (Ki = 2.7 KM)is the only other example of inhibition of a cysteine protease by a specific nitrile. This inhibition was completely reversible by dialysis. The fact that the ratio of Ki values (amidelnitrile) is roughly the same for 3b/3d as for 4b/4d also suggests that the mechanisms of inhibition are similar in both cases. One must now consider the possibility that inhibition of cysteine proteinases by specific nitriles may be a general phenomenon with potential for exploitation. Specific nitriles are reported not to be hydrolyzed by p a ~ a i n . ’ ~ JTo ~ rationalize their extraordinary potency, which is nearly as great as that of aldehyde analogues of specific ~ubstrates,’~ Lowe and Yuthavong13proposed that 8 bound to papain to give a Michaelis complex which is less strained than those for amides and peptides. Sluyterman and Wijdenes14 proposed that 8 might be a transition-state analogue for papain. Lewis and Wolfenden15considered that nitriles may bind covalently at the active site of papain by means of sulfhydryl addition (to form a thioimidate ester linkage). We are presently investigating the possibility of thioimidate bond formation by means of I3C NMR with papain and [nitrile-13C]-enriched 4d. By comparison to their effect on cysteine proteinases, specific nitriles are mediocre inhibitors of chymotrypsin and leucine aminopeptidase, i.e. the ratio of Ki values (amidelnitrile) is 2.6 for chymotrypsin, 0.22 for LAP-M, and 0.53 for LAP-C. Similar results were obtained for both LAP-M and LAP-C with L-LeuCONH, and L-LeuCN as inhibitors (unpublished results). Too little is yet known to speculate whether covalent intermediates are involved in the catalytic action of LAP, but the results with nitrile Id would not favor a covalent mechanism for the interaction of these enzymes with nitrile inhibitors. On the other hand, chymotrypsin is well known to form acyl enzyme intermediates, and yet nitrile 2d is hardly better than amide 2b as an inhibitor. This would appear to be consistent with the relatively greater nucleophilicity of the imidazolium-thiolate ion pair at the active site of papain16 (and perhaps DPP-I?) compared to the imidazole-hydrogen-bonded serine in chymotrypsin and with the recent suggestion by Asboth and Polgar” that electrophilic catalysis (by H-bond donors in the “oxyanion hole”) may not be as important with cysteine proteinases as it is with serine proteases (see below). Since nitriles have linear sp geometry, as opposed to the trigonal sp2 geometry of an

Thompson et al.

amide carbon, the nitrile N might not be in a suitable position to accept H bonds from the oxyanion hole of chymotrypsin. Indeed this is borne out by molecular graphics analysis (see below). Replacing the carbonyl oxygen of an amide or peptide unit by sulfur causes modest but well-characterized changes in chemical properties which can be useful for probing enzymic reaction mechanisms and/or creating enzyme inhibitors.l*J9 For example, X-ray studies have shown that except for the C=S bond being longer than C=O, there are no significant structural differences between oxopeptide and thiopeptide units.20 Another key difference is that C=S sulfur, being a soft Lewis base, is a poorer hydrogen-bond acceptor but binds to transition metal ions better than C=O oxygen.21 Recently several specific thioamides have been investigated as substrates and inhibitors of the zinc form of carboxypeptidase The thiopeptide bound as well as the oxopeptides (Le. K, = K,) but they hydrolyzed more slowly. The cadmium form of carboxypeptidase A, which does not hydrolyze peptides, was found to hydrolyze thiopeptide substrates.22 This finding was interpreted to support a role for metal ion coordination to the substrate during catalysis. In this context it is interesting to note that thioamide IC had no detectable inhibitory effect on LAP-C or LAP-M, both of which are zinc metalloenzymes. Likewise, L-leucinethioamide was also without effect on these enzymes (unpublished results). This could be taken to imply that direct zinc-substrate coordination is not involved in LAP action. Other evidence consonant with this view is the findingz3 that whereas LAP hydrolyzes only amino acid derivatives with unblocked amino termini, hydroxamic acid derivatives of D-Leu and even isocaproic acid (Le. desamino-Leu) inhibit LAP approximately as well as L-LeuCONHOH. On the other hand, it is possible that metal-substrate coordination does occur with L substrates, but that the D substrates and the desamino substrates bind from the S1’ position rather than S1. Lowe and Yuthavong found several thioamides to be competitive inhibitors of papain,13 although it was not reported whether they were hydrolyzed. Earlier, Lowe and Williams found that methyl hippurate and methyl thionhippurate were hydrolyzed by papain with nearly equal kcat/Kmvalues.24 More recently Asboth and Polgar recorded similar observations for pairs of ester/ thionoester analogues of specific substrates with papain.16 With chymotrypsin on the other hand, thionoester analogues of specific ester substrates are hydrolyzed extremely slowly, although they are bound quite well and competitively inhibit the hydrolysis of other substrates.”J8 Asboth and Polgar interpreted the differences between papain and chymotrypsin in behavior of ester/thionoester pairs of compounds to suggest that electrophilic catalysis (via an “oxyanion hole”) was important with serine proteases but perhaps not for cysteine proteases. Our results with 2b/2c and chymotrypsin are in accord with this. Although we did not determine if 2c was hydrolyzed by chymotrypsin, A.19322

Campbell, P.; Nashed, N. T. J . Am. Chem. SOC. 1982, 104,

5221. Bartlett, P. A.; Spear, K. L.; Jacobsen, N. E. Biochemistry

1982,21,1608. (12) Westerick, J. 0.;Wolfenden, R. J . Biol. Chem. 1974,249,6351.

(13) Lowe, G.;Yuthavong, Y. Biochem. J. 1971,124,107. (14) Sluyterman, L. A. Ae.; Wijdenes, J. Biochim. Biophys. Acta 1973,302, 95. (15) Lewis, C. A.; Wolfenden, R.Biochemistry 1977,16, 4890. (16) Polgar, L. Eur. J . Biochem. 1973,33,104. (17) Asboth, B.;Polgar, L.Biochemistry 1983,22,117.

LaCour, T.F. M.; Hansen, H. A. S.; Clausen, K.; Lawesson, S.-0. Znt. J . Peptide Protein Res. 1983,22,509. Hanzlik, R. P. “Inorganic Aspects of Biological and Organic Chemistry”;Academic Press: New York, 1976. Mock, W. L.; Chen, J.-T.: Tsang, J. W. Biochem. Biophys. Res. Commun. 1981,102,389. Wilkes. S.:Prescott. J. M. J . Biol. Chem. 1983,258, 13517. Lowe, G.; Wil1iams;A. Biochem. J . 1965,96,189.

Carboxyl-Modified Amino Acids and Peptides

Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1 107

Figure 1. Stereopair view of hemiacetal adduct of N-acetylphenylalaninalwith active site region of chymotrypsin. The phenyl of the inhibitor is in the center of the diagram, just below the aldehyde carbon (solid circle). The oxygen of Ser-195 is represented by a circle with a dot in the center. The OH of the hemiacetal is seen projecting toward the oxyanion hole (backbone NH of Ser-195 and Gly-193, which are represented by circles with crosses on them). The oxygen of the inhibitor acetyl group is hydrogen bonding (--) to the backbone NH of Gly-216. Compare to view of tosylchymotrypsin given in ref 25 and 27.

its Ki was within a factor of 2 of that of 2b. However with papain the Ki of thioamide 4c was considerably lower than that of amide 4b. Furthermore, although papain readily hydrolyzes methyl and ethyl t h i ~ n o h i p p u r a t ea, ~1~mM solution of thioamide 4c was essentially unaffected when incubated for 16 h at room temperature with 0.1-0.2 pM papain, as determined by monitoring the thioamide chromophore at 265 nm. Further detailed studies of the interaction of serine and cysteine proteinases with thioamides and thiopeptides would clearly be useful, both in connection with elucidation of enzyme mechanisms and to evaluate better the prospect of using thioamide bonds in designing protease inhibitors and/or metabolically stable peptides. The Michael acceptors e and f constitute a truly new group of protease inhibitors. Their design is based on the hope that the active-site nucleophile of a cysteine or serine protease might attack the /3-carbon of the ethylenic unit as if it were the carbonyl carbon of a normal substrate, leading to irreversible covalent inactivation of the enzyme (i.e. affinity labeling). Even though it is unclear, possibly even unlikely, that anything analogous to an acyl enzyme forms during LAP catalysis, le and If were tested against LAP. They were found to have very modest activity as competitive inhibitors, and no evidence for inhibition which increased with time was found. With chymotrypsin, 2e and 2f were found to be timeindependent competitive inhibitors with Ki values similar to that of thioamide 2c (i.e. roughly twice that of amide 2b). We interpret this observation to indicate that these compounds can at least bind to the active site of chymotrypsin and that the increased bulk of the Michael acceptor relative to a carbonyl did not severely restrict binding. Inspection of hypothetical CT-Michael acceptor Michaelis complexes by means of molecular graphics techniques supports the lack of steric inhibition of binding despite the presumed “tightness” of the oxyanion hole (see below). Thus we conclude that the failure of 2e or 2f to inhibit chymotrypsin irreversibly may be due to the failure of the chemical step of addition of the serine nucleophile to the polarized C=C bond. However, with papain and 4e this step evidently occurs quite readily, and 4e behaves like a true irreversible affinity-labeling reagent toward papain.3 This conclusion is supported by the following observations. The rate of papain inactivation by 4e is concentration dependent, and Kitz-Wilson analysis1° indicates rapid reversible E-I complex formation (Ki = 0.026 mM) followed by irreversible inactivation with first-order kinetics (k, = 0.0018 8-l). Thus the apparent second-order rate constant (k2/Ki)for inactivation of papain by 4e is 70 M-l s-l at 25 OC. Overnight dialysis of papain inactivated by

4e leads to no detectable reactivation, suggesting that the inactivation is indeed irreversible. The Michael acceptor moiety of 4e is absolutely essential for activity as shown by the relatively weak activity of 6, a des(methoxycarbony1) analogue of 4e, and 7, the saturated analogue of 4e, as competitive inhibitors of papain. Finally, the specificity of the inactivation process is reflected by the fact that incubation of papain with le (1mM for 20 min) led to no detectable loss of enzyme. Michael acceptors 3e and 3f also proved to be affinity labels for DPP-I. Although these compounds did not bind as well to DPP-I as 4e to papain, the irreversible reaction of their E-I complexes was faster (Table 11). Thus the apparent second-order rate constants ( k 2 / K i )for 3e and 3f were 68 and 55 M-l s-l, respectively, at 37 “C. The specificity of these processes is demonstrated by the fact that DPP-I showed no loss of activity after incubation for 90 min in a 1 mM solution of methyl trans-4-amino-2butenoate (5e). Molecular Graphics. In addition to biochemical evaluation of our compounds, the interactions of several of them with chymotrypsin and papain were examined by means of molecular graphics. Since this technique is relatively new, our main purpose was simply to determine the ways in which it might provide information complementary to or beyond that furnished by wet biochemical studies. We began by generating a stereoview of the active site of tosylchymotrypsin similar to that presented by Henderson,25using crystallographic coordinates provided by the Protein Data Bank.4 Standard bond lengths and angles were used for generating structures of inhibitors. The first inhibitor we examined by means of graphics was Nacetylphenylalaninal(9). This compound is thought to form a stable hemiacetal adduct with serine-195 of chymotrypsin. To fit this structure into CT, the hydrate of 9 (Le. N-Ac-PheCH(OH),, 10) was superimposed over the tosyl group by placing the aldehyde carbon at the locus of the tosyl sulfur, one of the oxygens of 10 over the oxygen of the tosyl group in the oxyanion hole, and the other oxygen of 10 essentially at the locus of the OH of serine195. The tosyl moiety was then eliminated entirely and the conformation of the remainder of molecule 10 was adjusted until a “good” visual fit was obtained (Figure 1). Attempts to incorporate (lO)-AcNH-O=C-Ser-214 hydrogen bonding, as discussed by various a ~ t h o r s , ~did ~-~’ (25) Henderson, R. J. Mol. BioE. 1970, 54, 341. (26) Steitz, T. A.; Henderson, R.; Blow, D. M. J. Mol. Biol. 1969, 46, 337. (27) Wipff, G.; Dearing, A.; Weiner, P. K.; Blaney, J. M.; Kollman, P. A. J. Am. Chem. SOC.1983,105, 997.

108 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1

Thompson et al.

Figure 2. Stereopair view of inhibitor 2e in the active site of chymotrypsin. The protein and the phenyl and acyl NH of the inhibitor are arranged as in Figure 1. The two solid circles represent the olefinic C=C bond of the inhibitor; other atoms are as in Figure 1.

Figure 3. Stereopair view of inhibitor 4d in the active site of papain. The sulfur of cys-25 is represented by a circle with a dot in its center, and the two olefinic carbons of the inhibitor are represented by solid circles. Compare to crystal structures of covalent papain-inhibitor complexes given in ref 29.

not lead to satisfactory geometries. However, we did find it possible to incorporate a Gly-218NH.-O=C(CH3)NH-( 10) hydrogen bond; this feature requires a cis amide bond in 10, but the overall complex has otherwise favorable geometry and intermolecular interactions. The CH(OH)2 group of the 10-CT complex was then replaced, without changing the coordinates of the remaining portion of 10, by the various groups b-f (see Scheme I); for 2b and 2d the carbonyl or thiocarbonyl groups were projected into the oxyanion hole. Visually it appeared that all of the groups, including the Michael acceptors e and f, fit well with no obvious steric problems. As an example, a potential Michaelis complex of 2e with chymotrypsin is depicted in Figure 2. Actual calculation of distances from the NH units of the oxyanion hole to various atoms of the groups b-f corroborated the conclusions drawn from visual inspection of the structures (data not shown). The examples shown in Figures 1and 2 do not represent the only binding modes accessible to inhibitors 2b-2f binding to chymotrypsin. Relaxation of the initial assumption that the carbonyl carbon equivalents of the inhibitors should be fixed at the same location as the tosyl sulfur in tosylchymotrypsin leads to an alternative binding mode similar to that proposed by Wipff et al.27for the with a-chymointeraction of D-N-acetyltryptophanamide trypsin. In this binding mode the inhibitors are capable of forming a hydrogen bond from the acyl nitrogen to the carbonyl group of Ser-214. Thus the major result of the molecular graphics studies with 2b-2f and chymotrypsin, i.e. that all compounds appear able to bind to chymotrypsin, agrees with the biochemical finding that all are at least moderately effective competitive inhibitors. The irreversible inhibition of cysteine proteases but not chymotrypsin by the Michael acceptors is undoubtedly due to the covalent interaction of the active site cysteine with these inhibitors having no counterpart in the case of chymotrypsin. Reversible covalent E-I interaction may account for the enormously greater potency of nitriles toward cysteine proteases as compared t o chymotrypsin,

as it does in the case of aldehydes.28 A similar type of analysis was carried out for papain starting with the model structure of the N-Cbz-L-Phe-LAla- acyl enzyme given by Drenth et al.4*29Inhibitor 4e was superimposed over the corresponding portions of the acyl enzyme structure, the Cbz-Phe-Ala moiety deleted, and the conformation of the ester group adjusted visually. The result is shown in Figure 3. The @-carbonof the Michael acceptor lies 180 pm directly above the sulfur of Cys-25; a normal C-S bond is ca. 172 pm. Thus the graphics supports the proposal, based on biochemical results, that 4e is a specific affinity label for papain. Experimental Section Melting points were determined in open capillary tubes and are uncorrected. NMR spectra were run on Varian FT-80, T-60, and EM-360 spectrometers with tetramethylsilane as an internal standard. Elemental analyses were performed on a HewlettPackard Model 185B instrument and are within f0.4% of the theoretical values. Optical rotations were measured with a Perkin-Elmer Model 141 polarimeter. Infrared spectra were taken on a Perkin-Elmer Model 33 spectrometer. Mass spectra were obtained on Ribermag R10-10 and Varian/Mat CH5 instruments by electron-impact ionization except where otherwise indicated. Spectrophotometric enzyme assays were performed on a Varian Cary 118 spectrophotometer. Mixed-Anhydride Couplings (Method A)! The N-protected amino acid was dissolved in THF and N-methylmorpholine (1 equiv) was added. The solution was cooled to -15 "C, ethyl chloroformate (1equiv) was added, and the solution was stirred for 20 min. The amine was dissolved in THF with N-methylmorpholine (1equiv) and added to the reaction mixture while a temperature of -15 "C was maintained. Stirring was continued for 30 min at this temperature and a further 1-2 h while the reaction was allowed to warm to room temperature, after which ~~~~~

(28) Gamcsik, M. P.; Malthouse, J. P. G.; Primrose, W. U.; Mackenzie, N. E.; Boyd, A. S. F.; Russell, R. A.; Scott, A. 1. J. Am. Chem. Soc. 1983, 105, 6324. (29) Drenth, J.; Kalk, K. H.; Swen, H.M.Biochemistry 1976,15, 3731.

Carboxyl-Modified Amino Acids and Peptides the solvent was removed under reduced pressure. The residue was dissolved in CH2C12,washed with 2% HCl and saturated NaHC03, and dried (MgS04). The solvent was removed under reduced pressure to give the crude product. Amine Protection and Deprotection (Methods B and C). The amino acid was dissolved in dioxane/H20 (2:l)with 1 N NaOH (1equiv). Di-tert-butyl pyrocarbonate (1equiv) was added and the solution was stirred overnight while C02 evolved. The dioxane was removed under reduced pressure and the residue was extracted into CHCl,. The organic solution was washed with 2% HCl and dried (MgSO,), and the residue was purified by chromatography or crystallization. For removal of the tert-butoxycarbonyl group, the N-Boc compound was dissolved in trifluoroacetic acid and allowed to stand for 20 min. The trifluoroacetic acid was removed under reduced pressure and the trifluoroacetate salt was purified by crystallization. In cases where the TFA salt was hygroscopic, the crude product was dissolved in methanol, oxalic acid (1 equiv) or methanesulfonic acid (1 equiv) was added, and the solvent was removed in vacuo. Methanol was added three more times and removed in vacuo each time. The white product was purified by crystallization. Acetylation of Amines (Method D). The amine compound was dissolved in THF, Et3N (2equiv) added, the solution cooled to 4 "C, and acetic anhydride (2equiv) added. The solution was allowed to warm to room temperature (45 min) and the solvent removed in vacuo. The residue was dissolved in CHC1, and washed with 2% HC1 and saturated NaHC03. After filtration and evaporation, the product was purified by crystallization. Horner-Wadsworth-Emmons Reaction (Method E). NaH (50% in mineral oil) was washed twice with hexane, and THF was added. Trimethyl phosphonoacetate (or dimethyl [(methylsulfonyl)methyl]phosphonate)(1 equiv) was added and the solution stirred for 30 min while H2 evolved. The aldehyde (1 equiv) was dissolved in THF and added to the reaction mixture, and the mixture was stirred for 1.5 h. The reaction was quenched with H20 and the THF was removed under reduced pressure. The residue was dissolved in CHC13,washed with 2% HCl and saturated NaHC03, dried (MgSO,), and evaporated to a yellow product, which was purified by chromatography or crystallizatioh. L-Phenylalanine Nitrile Methanesulfonate (ld). N-Bocwas dissolved in dry THF Gphenylalaninamide (1.31g, 4.97 "01) (50 mL) and Et3N (1.5 mL, 2.2equiv) was added. The solution was cooled on an ice bath to 5 "C and trifluoroacetic anhydride (0.78mL, 1.1 equiv), was added over 5 min. The solution was allowed to warm to room temperature. After 30 min, the reaction was quenched with H 2 0 (3mL), the THF removed in vacuo, and the product extracted into ether. The ether was washed with dilute acid and base and dried (MgSO,). The solvent was removed and the crude product (11)was purified (1.15g, 94%) by silica gel chromatography (EtOAc-hexane): mp 117.5 "C; NMR (CDC13) 6 1.43 (s, 9), 3.07 (t, 2), 4.60-4.85 (br s, l), 7.31 (s, 5); CI mass spectrum, m/e (relative intensity) 247 (34,M + 1) 208 (51),191 (47),163 (12),120 (96),91 (100). Anal. (C14HlsN202)C, H, N. Compound 11 (500mg, 2.03 mmol) was deprotected (method C). The residue was dissolved in EtOH (10mL), methanesulfonic acid (0.13mL, 1 equiv) was added, and the solvent was evaporated. The crude product was crystallized from EtOH (10mL) and E t 0 (90 mL), yielding Id (250mg, 52%): mp 162.5-163 'C dec; NMR (D20)6 2.70 (s, 3,CH3S03),3.25 (d, 2,J = 8 Hz), 4.58-4.82 (m, 2), 7.35 (s, 5). Anal. (CloHI4N2O3S)C, H, N. N-Acetyl-L-phenylalanineNitrile (2d). Compound Id (288 mg, 1.22"01) was dissolved in dry THF (15mL) and acetylated (method D). The crude product was recrystallized from CHC13 (10 mL) and hexane (140 mL), yielding 160 mg (70%): mp 125-126 "C; NMR (CDC13) 6 1.99 (9, 3),3.08 (d, 2, J = 6 Hz), 4.98-5.27 (m, l), 5.69-5.94 (br s, l),7.33 (9, 5); mass spectrum, m/e (relative intensity), 188 (6,M'), 162 (l), 144 (l), 129 (58), 91 (loo), 65 (23). Anal. (CllH12N20)C, H, N. Glycyl-L-phenylalanine Nitrile Methanesulfonate (3d). Boc-glycine (232mg, 1.33 mmol) was coupled to compound Id (1.33 mmol) by method A. The crude product (12) was recrystallized from CH2C12(10mL) and hexane (90mL), which yielded 346 mg (86%): mp 100-100.5 "C; NMR (CDCl,) 6 1.44(s, 9),3.07 (d, 2,J = 7 Hz), 3.74 (d, 2,J = 6 Hz), 4.90-5.20 (m, 2), 7.00 (d, 1, J = 8 Hz), 7.31 (s, 5);mass spectrum, m/e (relative intensity)

Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1 109

247 (13),230 (22),220 (9),203 (6),229 (71),120 (9),91 (62),57 (100). Anal. (C16H22N303) C, H, N. Compound 12 (343mg, 1.13 mmol) was deblocked by method C. The crude product was dissolved in MeOH and 1 equiv of methanesulfonic acid was added. The solution was concentrated and the crude solid was recrystallized twice from hot EtOH to give 125 mg of 3d (37% yield): mp 234.5-235 'C; NMR (D2O) 6 2.74 (9, 3,CHpSO,), 3.17 (d, 2,J = 8 Hz), 3.73 (9, 2), 5.05 (t, 1, J = 8 Hz), 7.34 (s,5);mass spectrum, m/e (relative intensity) 204 (5,M+),159 (28),129 (17),104 (lo),91 (loo),77 (18),65 (31). Anal. (Ci2Hi7N304S) C, H, N. [N-(N-Acetyl-L-phenylalaninyl)amino]acetonitrile (4d). N-Acetylphenylalanine (5 g, 24.15 mmol) was coupled to aminoacetonitrile hydrochloride (2.23g, 24.15 mmol) by method A. (Later experience showed that DMF was much preferred over THF or CH,Cl2 for this reaction.) Recrystallization from EtOH (100mL), CHC13 (50 mL), and hexane (700 mL) gave 3.33 g of product (56% yield): mp 183.5185.5"C; NMR (MeOH-d,) 6 1.68 (s,3),2.48-2.96 (m,2),3.86 (s,2),4.23-4.43 (m, l),7.01 (s,5, Ar); mass spectrum, m/e (relative intensity) 2.45 (4,M'), 186 (31), 162 (7),131 (31),120 (loo),112 (24),103 (27),91 (loo), 77 (27). Anal. (Ci3H15N302)C, H, N. Methyl 4-Amino-5-phenyl-2-pentenoate Trifluoroacetate (le). Boc-phenylalaninal(l3) was prepared from Boc-Phe-OMe as described by Rich et al. for Boc-~-leucinal.' The ester (10.6 g) was dissolved in dry toluene (200mL) and cooled to -78 "C. Dibal-H (1 M in hexane, Aldrich, 75.5 mL) was added over 35 min and the solution was stirred. After 5 min the reaction was quenched with MeOH (3.2mL) and allowed to warm to room temperature. The solvent was removed in vacuo and the residue was dissolved in E t 2 0 (1 L) and washed with saturated NaK tartrate solution. The ether was reduced to 200 mL, washed with dilute acid and base, dried (MgSO,), and removed in vacuo, yielding a solid material. This was dissolved in 20 mL of EtOH and added dropwise to a cold solution of 1 N NaHSO, (300mL) and allowed to stir overnight at 4 "C. The resulting solution was washed with CHC1, (200 mL), made alkaline with K2CO3, and extracted with CHC13 (600mL). The CHCl, solution was dried (MgSOJ and the solvent was evaporated to give 5.31 g (56%) of a white solid, Boc-phenylalaninal: mp 86 "C; [ C Y ] ~-D42.5' (c 2.4, MeOH); NMR (CDC13)6 1.36 (s, 9),2.86-3.03 (m, 2), 4.22 (m, l), 5.26 (d, 1,J = 7 Hz), 7.23 (s,5),9.40 (s,l); mass spectrum, m/e (relative intensity) 200 (23),164 (54),120 (83),91 (19),57 (100). Anal. (Cl4HlsNO3)C, H, N. Boc-phenylalaninal (2.49 g, 10 mmol) was condensed with trimethyl phosphonoacetate by method E. The product (14)was obtained by recrystallization from EtOH and H 2 0 (1:l)(200mL) 0.52' (c 1.1,CH2C12); to give 2.01 g (66.5%): mp 68-70 'C; NMR (CDC13) 6 1.39 (s, 9), 2.88 (d, 2, J = 7 Hz), 3.72 (s, 3), 4.35-4.75 (br, 2), 5.85 (d, 1,J = 16 Hz), 6.9 (dd, 1,J = 16,5 Hz), 7.26 (s, 5); mass spectrum, m/e (relative intensity) 305 (1,M+), 214 (31),158 (45),114(78),91 (24),57 (100). Anal. (C17H2,N04) C, H, N. Compound 14 (2 g, 6.56 mmol) was deprotected with trifluoroacetic acid (15 mL) by method C. The product (le) was recrystallized from CHC13 (3mL) and E t 0 (40mL): yield 1.85 g (88%);mp 137.5-138 'C dec; [ff]=D 45.5"(c 1.0,CH30H); NMR (D2O) 6 3.04 (d, 2,J = 7 Hz), 3.67 (5, 3), 4.23 (d, 1, J = 7 Hz), 5.88 (d, 1,J = 16 Hz), 6.68 (dd, 1,J = 16,7 Hz), 7.28 (s, 5);mass spectrum, m/e (relative intensity), 206 (l), 174 (3),144 (l), 128 (11,114 (loo), 91 (141,54 (24). Anal. (C14H17F3N04) C, H, N. Methyl N-Acetyl-4-amino-5-phenyl-2-pentenoate (2e). Compound l e (490 mg, 1.54 mmol) was acetylated with acetic anhydride (2 equiv) by method D. The crude product was recrystallized (284mg, 75%) frm CHC13-hexane (1:20): mp 83-83.5 'C; NMR (CDC13) 6 1.94 (5, 3), 2.91 (d, 2,J = 7 Hz), 3.71 (5, 3), 4.95 (br, l), 5.40 (br, 11, 5.82 (d, 1, J = 16 Hz), 6.93 (dd, 1, J = 16,6Hz), 7.25 (8, 5); mass spectrum, m/e (relative intensity) 247 (1,M'), 204 (l),188 (6),174 (8),156 (43,129 (lo),114 (loo),91 (30),65 (13),54 (17),43 (46). Anal. (C14H17N03)C, H, N. Methyl N-Glycyl-4-amino-5-phenyl-2-pentenoate Oxalate (3e). Boc-glycine (87 mg, 0.5 mmol) was coupled to methyl 4-amino-5-phenyl-2-pentenoate by method A. The crude product (15) was purified by column chromatography (EtOAc-hexane) which gave 126 mg (70%) of a very viscous oil: NMR (CDCl3) 6 1.45 ( 8 , 9),2.90 (d, 2,J = 7 Hz), 3.70 (s, 31,3.73 (d, 2,J = 6 Hz),

110 Journal of Medicinal Chemistry, 1986, Vol. 29, No. 1

Thompson et al.

(d, 1, J = 15 Hz), 6.83 (dd, 1, J = 15, 6 Hz), 7.26 (s, 5 ) ; mass 4.90 (m, l),5.52 (t, 1, J = 6 Hz), 5.80 (dd, 1,J = 16, 2 Hz), 7.24 spectrum, m / e (relative intensity) 268 (2, M + l),208 (6), 188 (s, 5), 6.70-7.32 (m, 2); mass spectrum, m / e (relative intensity) (16), 176 (12), 146 (121, 134 (801, 129 (20), 128 (23), 91 (70), 65 363 (1,M + l), 306 (l),289 (l), 271 (71,215 (16), 197 (5), 188 (7), 174 (5), 171 (7), 129 (15), 114 (loo), 91 (27), 84 (44), 57 (51). (26), 54 (63), 43 (100). Anal. (C13H17N03S)C, H, N. Boc-glycine (291 mg, 1.66 mmol) was coupled with If (563 mg, Compound 15 (279 mg, 0.77 mmol) was deprotected with 1.66 mmol) by method A. The product (17) was recrystallized trifluoroacetic acid (3 mL) by method C. The crude product was from EtOAc (18 mL) and hexane (80 mL): yield 476 mg (75%); dissolved in MeOH (5 mL) and oxalic acid (107 mg, 0.85 mmol) mp 91 OC; NMR (CDCI,) 6 1.42 (s, 9), 285 (s, 3), 2.91 (d, 2, J = was added. The solvent was removed in vacuo. MeOH was added 7 Hz), 3.69 (d, 2, J = 6 Hz), 4.78-5.13 (br, l),5.43 (t, 1,J = 6 Hz), and removed in vacuo a total of four times to remove residual 6.39 (d, 1, J = 15 Hz), 7.20 (s,5), 6.68-7.3 (m, 2); mass spectrum, trifluoroacetic acid. The crude product (185 mg, 66%) was m / e (relative intensity) 309 (l),303 (2), 247 (3), 235 (5), 155 (lo), precipitated from MeOH (8 mL) and EhO (100 mL): mp 172 "C; 134 (24), 91 (221, 57 (100). Anal. (Cl8HZ8N2O5S) NMR (D20)6 2.73-3.02 (m, 2), 3.65 (s, 5), 4.58-4.88 (br, l),5.84 C, H, N. Compound 17 (44.7 mg, 0.12 "01) was deprotected by method (dd, 1,J = 16, 2 Hz), 6.91 (dd, 1, J = 16, 5 Hz), 7.24 (s, 5); mass C and the counterion was changed to oxalate as with compound spectrum, m / e (relative intensity) 263 (1, M + l),262 (1, M+) 3f. The product was precipitated cleanly (40.7 mg, 91%) from 245 (l), 233 (l), 214 (3), 188 (6), 171 (60), 139 (8), 129 (23), 114 (loo), 91 (49), 82 (ll),65 (15). Anal. (C16HzoN207) warm MeOH (10 mL) and EhO (10 mL): mp 198-199 OC; NMR C, H, N. (D20)6 2.87 (d, 2, J = 8 Hz), 2.97 (s,3), 3.65 (s, 3),4.60-5.00 (br, Methyl 4-Amino-2-butenoate Trifluoroacetate (5e). NI), 6.53 (d, 1, J = 16 Hz), 6.93 (dd, 1, J = 16, 4 Hz), 7.25 (s, 5); (tert-Butoxycarbonyl)-3-aminopropene (2.13 g, 13.57 mmol) was mass spectrum, m / e (reltive intensity) 283 (3, M + l), 282 (3, M+), dissolved in 50 mL of MeOH and the mixture cooled to -78 "C. 203 (84), 246 (25), 134 (49), 120 (70), 111 (loo), 91 (loo), 83 (65), Ozone was bubbled through for 20 min (until the solution became 65 (74), 56 (85). Anal. (C15H2,,N207S) C, H, N. blue). Nitrogen was passed through the solution to displace the N-Glycyl-L-phenylalaninamide Methanesulfonate (3b). excess ozone and dimethyl sulfide (1.5 mL, 20.4 mmol) was then N-Boc-glycine (363 mg, 2.07 mmol) was coupled to phenylalanadded. The solution was allowed to warm to room temperature inamide hydrobromide (508 mg, 2.07 mmol) by method A. The very slowly (6 h or more). The solution was concentrated and product 18 was recrystalliied from EtOH (2 mL), CHC1, (20 mL), dissolved in CHC13, washed with dilute acid and base, and dried and hexane (50 mL): yield 518 mg (78%);mp 167-168 "C; NMR (MgS04). Evaporation gave 2.04 g (94%) and the product, N(CDCl,) 6 1.41 (s,9), 3.05-3.16 (m, 2), 3.73 (d, 2, J = 5.8 Hz), 4.70 (tert-butoxycarbonyl)glycinal,was used without further purifi(q,l,J=7.2Hz),5.14(br,t,l,J=5.6Hz),5.53(br,s,1),6.14 cation. It was condensed with trimethyl phosphonoacetate (1 (br, s, l),6.79 (br, d, 1,J = 7.8 Hz), 7.26 (s, 5). Anal. (c16H&304) equiv) by method E to produce the Boc derivative of 5e. This C, H, N. compound was purified by medium pressure silica gel chromaThe product of the above reaction was deprotected by method tography (EtOAc-hexane): yield 280 mg (62%); mp 45 "C; NMR C and the counterion was changed to mesylate by the method (CDC13) 6 1.44 (s, 9), 3.69 ( 8 , 3), 3.73-3.96 (m, 2), 6.63 (br s, l), used for compound Id. The product was recrystallized from 5.87 (d, 1,J = 16 Hz), 6.88 (dt, 1 J = 16, 5 Hz); mass spectrum, MeOH (60 mL) and E t 2 0 (60 mL): yield 359 mg (90%); mp m / e (relative intensity) 159 (23), 142 (E),127 (lo), 115 (8), 84 199-201 OC; NMR (DzO) 6 2.72 (s, 3), 2.93-3.09 (m, 2), 3.69 (s, (31), 57 (100). Anal. (C10H1,N04)C, H, N. 2), 4.49-4.63 (m, l),7.28 (s, 1). Anal. (Cl2Hl,N3O5S) C, H, N. Boc-5e (327 mg,1.52 "01) was deprotected by method C. The N-(N-Acetyl-L-phenylalaniny1)glycinamide(4b). Ncrude product was recrystallized from MeOH (1mL) and EtzO Acetyl-L-phenylalanine(1.0 g, 4.83 mmol) was coupled to methyl (10 mL), yielding 327 mg of 5e (94%): mp 120-120.5 "C; NMR glycinate hydrochloride (0.607 g, 4.83 mmol) by method A. The (acetone-&) 6 3.69 (s, 3), 3.91 (d, 2, J = 6 Hz), 4.59 (d, 3, J = 6 product (19) was precipitated from CHzClz(50 mL) with hexane Hz), 6.09 (dd, 1, J = 16, 8 Hz), 6.91 (dt, 1, J = 16, 6 Hz). Anal. (170 mL): yield 851 mg (63%); mp 137-138.5 "C; NMR (CDC13) ( C ~ H ~ O F ~ C, NO H,~ N. ) Methyl N-(N-Acetyl-~-phenylalanyl)-4-amino-2-pent- 6 1.91 (s, 3), 3.03 (d, 2, J = 7 Hz), 3.68 (s, 3), 3.93 (d, 2, J = 6 Hz), 4.80 (q, 1,J = 7 Hz), 6.55-7.10 (m, 2), 7.23 (s, 5); mass spectrum, enoate (4e). N-Acetyl-L-phenylalanine(501 mg, 2.42 mmol) was m / e (relative intensity) 2.78 (2, M'), 247 (l),219 (16), 190 (6), coupled to compound 5e by method A. Precipitation from EtOH 162 (16), 145 (33), 131 (271,120 (loo),91 (25). Anal. (C14H18Nz04) (5 mL) and CHC13 (25 mL) with hexane (400 mL) yielded pure C, H, N. 4e (70%): mp 156.5-157 "C; NMR (CDC13)6 1.95 (s,3), 3.05 (d, Compound 19 (315 mg, 1.13 mmol) was dissolved in MeOH (10 2, J = 8 Hz), 3.70 (s, 3), 3.80-3.97 (m, 2), 4.66 (q, 1,J = 8 Hz), mL) and cooled to 5 "C. NHBwas bubbled through the solution 5.66 (d, 1, J = 16 Hz), 6.69 (dd, 1, J = 16, 6 Hz), 6.20-6.60 (br, for 2.5 h. The solution was allowed to warm to room temperature m, 2), 7.2 (s,5); mass spectrum, m / e (relative intensity) 304 (4, and stirred for 8 h. The solvent was evaporated and the white M+), 261 (3), 245 (4), 213 (2), 190 (5), 162 (22), 120 (100),114 (261, product was recrystallized (239 mg, 81%) from MeOH (4 mL), 91 (26). Anal. (C16HzoNz04) C, H, N. EtOAc (20 mL), and EkO (2 mL): mp 184 "C; NMR (CDSOD) 3-Amino-4-phenyl-l-(methylsulfonyl)-l-butene Tri6 1.70 (s, 3), 2.80 (t, 2), 3.55 (d, 2, J = 6 Hz), 4.25 (dd, 1, J = 8, fluoroacetate (If). Boc-phenylalaninal(l3) was condensed with 7 Hz), 7.00 (s, 5); CI mass spectrum, m / e (relative intensity) 264 dimethyl [(methylsulfonyl)methyl]phosphonate(6.3 g, 31.2 "01) (36, M + I), 247 (lo), 204 (ll),190 (43), 162 (35), 146 (68), 131 by method E. The product (16)was recrystallized from CHC1, (27), 120 (loo), 91 (27). Anal. (C13H17N303)C, H, N. (30 mL) and hexane (100 mL): yield 1.25 g (58%); mp 143-144 L-Phenylalanine Thioamide (IC). Boc-L-phenylalaninamide 'C; [ c Y ] ~ ~6.68" D ( C 0.9, CH2C12); NMR (CDC13) 6 1.36 (9, 9), 2.83 was dissolved in HMPA (20 mL). Lawesson's (4.0 g, 15.15 "01) (s, 3), 2.93-3.02 (d, 2, J = 4 Hz), 4.66 (br, l), 6.06-5.52 (br, l), reagent6 (Aldrich, 3.1 g, 7.6 mmol) was added and the solution 6.30 (d, 1, J = 16 Hz), 6.9 (br d, 1,J = 16 Hz), 7.27 (s, 5); mass was stirred at 80 OC for 1 h. The solution was allowed to cool spectrum, m / e (relative intensity) 270 (8), 252 (12), 246 (15), 234 to room temperature and the reaction mixture was partially (E),190 (60), 178 (21), 134 (92), 91 (100). Anal. (Cl6H&o4S) purified by silica gel chromatography (EtOAc, hexane). The C, H, N. product (20) was twice recrystallized from EtzO (10 mL) and Compound 16 (502 mg, 1.54 mmol) was deprotected with hexane (500 mL) to yield 2.95 g (70%): mp 104-105 "C; NMR trifluoroacetic acid (6 mL) by method C. The product (If) was (CDC1,) 6 1.33 (s, 91, 3.07 (d, 2, J = 8 Hz), 4.70 (4, 1,J = 8 Hz), recrystallized from MeOH (10 mL) and EhO (50mL): yield 480 5.53 (d, 1, J = 8 Hz), 7.27 (s,5), 7.77-8.07 (br 2); mass spectrum, mg (92%); mp 196-200 OC dec; [ ~ t33.9" ] ~ (c 1.0, ~ MeOH); NMR m / e (relative intensity) 280 (4, M+), 224 (13), 220 (8), 207 (5), (D,O) 6 2.95 (s, 3), 2.98-3.23 (m, 2), 4.15-4.47 (m, l),6.55 (d, 1, 163 (32), 130 (8), 120 (68), 91 (54), 57 (100). Anal. ( C ~ ~ H ~ $ J Z O ~ S ) J = 16 Hz), 6.82 (dd, 1,J = 16, 6 Hz), 7.3 (s, 5); mass spectrum, C, H, N. m / e (relative intensity) 226 (l),146 (12), 134 (56), 91 (31), 72 (20), Compound 20 (201 mg, 0.72 "01) was deprotected by method 54 (100). Anal. (C13H16F3N04S)C, H, N. N-Acetyl-3-amino-l-(methylsulfonyl)-4-phenyl-l-butene C. The product was recrystallized from EtOH (1mL), Et20 (5 mL), and hexane (30 mL) to yield 197 mg (93%): mp 168-169.5 (2f). Compound If (410 mg, 1.21 mmol) was treated with acetic 6 3.17 (d, 2, J = 7.4 Hz), 4.37 (t, 1,J = 7.4 "C dec; NMR (DzO) anhydride (2 equiv) by method D. The product was recrystallized Hz), 7.31 (s, 5); mass spectrum, n / e (relative intensity) 180 (4, from EtOH (10 mL) and hexane (50 mL): yield 291 mg (90%); M+), 163 (40), 130 (7), 120 (loo), 104 (19), 91 (36). Anal. (Cllmp 164 OC; NMR (CDClJ 6 1.96 (s, 31, 2.88 (s, 3), 2.95 (d, 2, J = 6 Hz), 4.94 (br t, 1, J = 6 Hz), 5.57 (br d, 1,J = 6 Hz), 6.78 HI~F~NZOZ C,SH, ) N.

Carboxyl-Modified Amino Acids and Peptides

N-Acetyl-L-phenylalanine Thioamide (2c). Compound IC (112 mg, 0.38 mmol) was acetylated by method D. The product was recrystallized from CHC1, (10 mL) and hexane (50 mL): yield 64 mg (76%); mp 163-164 "C; NMR (CDSOD) 6 1.66 ( 8 , 31, 2.73-2.97 (m, 2), 4.45-4.73 (m, l), 7.02 (8, 5); mass spectrum, mfe (relative intensity) 222 (14, M+), 162 (35), 129 (43), 120 (72), 91 (loo), 89 (300), 77 (14),65 (26). Anal. (CllH14Nz0S) C, H, N. N-(N-Acetyl-L-phenylalaniny1)glycine Thioamide (412). Compound 4d (1.25 g, 5.10 mmol) was dissolved in 15 mL of pyridine and 10 mL of Et,N with mild heating. HzS was bubbled through for 30 min and the solution was allowed to stir overnight. The solution was diluted with 300 mL of ether and a precipitate separated. The supernatant was evaporated and extracted into EtOAc. The solution was washed with dilute acid and base and dried (MgS04). The solvent was removed and the product was recrystallized from EtOH (10 mL), EtOAc (10 mL), and hexane (400 mL): yield 441 mg (38%); mp 178 OC; NMR (MeOH-d,) 6 1.68 (s, 3), 2.60-2.95 (m, 2), 3.86 (d, 2, J = 6 Hz), 4.06-4.30 (m, l),6.98 (s, 5); mass spectrum, m f e (relative intensity) 279 (10, M+) 220 (17), 162 (16), 132 (22), 120 (loo), 91 (63). Anal. (Ci&i$J302S) C,H, N. N - (N-Acetyl-~-phenylalaninyl)-3-aminopropene (6). N-Acetyl-L-phenylalanine(2.5 g, 12.08 mmol) was coupled to allylamine (0.91 mL, 12.08 mmol) by method A. The product was crystallized to fine white needles from hot EtOH (20 mL) and hexane (200 mL): yield 957 mg (32%); mp 160-161 "C; NMR (CDC1,) 6 1.96 (s, 3), 3.06 (d, 2, J = 6.7 Hz), 3.75 (t, 2, J = 5.5 Hz), 4.65 (9, 1,J = 7 Hz), 4.80-5.15 (m, 2), 5.40-6.00 (m, l ) , 6.00 (br, l), 6.38 (br d, 1,J = 7 Hz), 7.24 (s, 5). Anal. (C14H18N202) C, H, N. Methyl N -(N-Acetyl-~phenylalainyl)-4-aminobut~oate (7). N-Acetyl-L-phenylalanine(0.5g, 2.42 mmol) was coupled to methyl 4-aminobutanoate (370 mg, 2.42 mmol) by method A. The product was crystallized from CHCI, (5 mL), EhO (60 mL), and hexane (20 mL): yield 276 mg (37%); mp 120-120.5 "C; NMR (CDC13) 6 1.56-1.75 (m, 2), 1.97 (s, 3), 2.11-2.28 (m, 2), 2.97-3.21 (m, 4), 3.65 (8, 3), 4.51 (4, 1, J = 6.4 Hz), 5.88-6.43 (br, 2), 7.23 (s, 5). Anal. (Cl6HzZNzO4) C, H, N. Assay and Inhibition of Chymotrypsin. Chymotrypsin (Worthington, crystallized three times) was assayed with use of N-glutaryl-L-phenylalanine p-nitroanilide essentially as described by Erlanger et al.,30 except that the rate of substrate hydrolysis at 25 "C was monitored continuously at 410 nm (Ac = 8600 M-' cm-'). Test compounds were added in small aliquots of methanol (